Alterations in the stiffness of lipid bilayers are likely to constitute a general mechanism for modulation of membrane protein function. Gramicidin channels can be used as molecular force transducers to measure such changes in bilayer stiffness. As an application, we show that N-type calcium channel inactivation is shifted reversibly toward negative potentials by synthetic detergents that decrease bilayer stiffness. Cholesterol, which increases bilayer stiffness, shifts channel inactivation toward positive potentials. The voltage activation of the calcium channels is unaffected by the changes in stiffness. Changes in bilayer stiffness can be predicted from the molecular shapes of membrane-active compounds, which suggests a basis for the pharmacological effects of such compounds.